Enzyme electrode based biosensors cover a broad application field, including clinical diagnostics, food analysis and environmental monitoring and control. Since 1962 when the first glucose sensor was developed by Clark and Lyons, over 100 enzyme electrode based biosensor tests have been developed and made commercially available. (Phil. Trans. R. Soc. Lond. B 316, 85-94, 1987). By definition, biosensors are devices comprising an analyte and a selective interface in close proximity or integrated with a transducer, which relays an interaction between the interface and the analyte, directly or through a mediator. The analyte interface is a bioactive substance, e.g., an enzyme, antibody, or micro-organism, etc., that is capable of recognizing cognate analytes and configured to regulate the specificity and sensitivity of the device. The transducer converts the biochemical signal into an electrical signal, which can be suitably processed and output.
Electrochemical biosensors are a common class of enzyme electrode based biosensors, and are generally based on the fact that during the bio-interaction process, electrochemical species such as electrons are consumed or generated producing an electrochemical signal that can be measured by an electrochemical detector. An amperometric enzyme electrode is one type of an electrochemical biosensor that has been used widely as it is capable of directly transducing the rate of an enzyme-catalyzed reaction into a current. More specifically, amperometric biosensors function by the production of a current when a potential is applied between two electrodes.
The bioactive substances found in amperometric enzyme electrodes commonly comprise redox enzymes that oxidize substrates by accepting and transferring electrons to electron acceptors (e.g., O2). Amperometric enzyme electrodes are configured to measure a current proportional to the concentration of O2 or the rate of production of the product H2O2. However, such biosensors may be dependent on the concentration of dissolved O2 in the sample. For example, the rate of electrochemical reduction of O2 depends on the rate of diffusion of oxygen from the bulk solution, which in turn is dependent on the concentration gradient and hence the bulk oxygen concentration. Thus, such biosensors are dependent on the concentration of dissolved oxygen in the bulk solution.
In order to overcome these problems, the concept of using artificial electron acceptors evolved to avoid the reduction of oxygen. In these biosensors, all the substances having conversion potential lower than the electrode potentials contribute to the overall electrochemical signal. The most common example is that at the oxidation potential of +600 mV along with H2O2 other metabolites such as uric acid, ascorbic acid, glutathione, etc., also get oxidized and interfere with the electrochemical signal. It is therefore, important to apply an electrode potential as low as possible. In order to achieve a low electrode potential, electrochemically active electron acceptors to which the enzyme can donate electrons were investigated. In this context, some artificial electron acceptors having low oxidation potentials were discovered. These artificial electron acceptors are commonly called mediators. This approach leads to a considerable reduction of electrochemical interferences and the development of mediated biosensors. Thus, mediated biosensors can be constructed with the enzymes that can donate electrons to electrochemically active artificial electron acceptors.
Mediated biosensors typically employ a two step procedure in which the enzyme takes part in first a redox reaction with the substrate and is in turn reoxidized by a mediator and finally the mediator is oxidized by the electrode. The amperometric procedure leads to the utilization of a lower redox potential that can be used for H2O2 detection. If the fixed concentration of an electron acceptor is retained within the enzyme layer, the operational stability of the sensor can be increased. The amperometric biosensors incorporating immobilized mediators therefore provide an effective alternative to peroxide detecting systems.
Mediators are artificial electron transferring agents that can readily participate in the redox reaction with the biological component and thus help in rapid electron transfer. It is a low molecular weight redox couple, which shuttles electrons from the redox center of the enzyme to the surface of the indicator electrode. During the catalytic reaction, the mediator first reacts with the reduced enzyme and then diffuses to the electrode surface to undergo rapid electron transfer. The rate of production of the reduced mediator is measured amperometrically by oxidation at the electrode. Advantageously, mediated enzyme electrodes are known to be less susceptible to interfering substances due to lower electrode potentials.
In 1984, Cass et al. developed a biosensor using a ferrocene mediator for a glucose sensor design (Cass et al., Anal. Chem., 1984, 56, 667), which enabled lower potential detection with less interference. Therefore, choosing suitable mediators is another key component for mediator-enzyme electrode design. The most commonly used mediators are Ferrocyanide or Ferrocene and Quinone deriviatives. Osmium complexes, polypyrrole, organic dyes are also mediators considered under certain circumstances.
The typical mediator-enzyme coupling sensor involves either covalently attaching the mediator to the enzyme or enzyme immobilization. Previous studies have focused on covalently attaching different mediators to the backbone of enzymes to enhance electron transfer directly to the electrode. (Heller et al., J. Am. Chem. Soc., 1988, 110, 2615; J. Am. Chem. Soc., 1991, 113, 1394; J. Phys. Chem., 1992, 96, 3579; Bartlett et al., Talanta, 1991, 38, 57; J. Chem. Soc. Chem. Commun., 1987, 103). However, the studies observed poor stability of the sensor due to mediator decomposition. Different mediator derivatives also play important impacts on the sensor performance. As for immobilized enzymes on the electrode, researchers have tried different materials including hydrogels (Updike et al., Nature, 1967, 214, 986; Guilbault et al., J. Am. Chem. Soc., 1969, 91, 2164; 1970, 92, 2533; Anal. Chem., 1974, 46, 1769), conducting polymers (Foulds et al., J. Chem. Soc. Faraday trans., 1986, 82, 1259; Waller et al., J. Anal. Chem., 1986, 58, 2979; Yabuki et al., J. Chem. Soc. Chem. Commun., 1989, 945; Bartlett et al., J. Electroanal. Chem., 1987, 224, 37; J. Chem. Soc. Faraday trans, 1992, 88, 2677), non-conducting polymers (Burno et al., J. Electrochim. Acta, 1977, 22, 451) and silane modified surfaces (Weetall et al., 1988) in analytical uses of immobilized biological compounds for detection, medical and industrial uses. However, this approach is associated with higher background and interference problems. It also requires efforts on studying and evaluating of different materials for immobilizing or entrapping enzymes on the sensor.
While many native enzymes can be used in electrochemical biosensors using mediators, several enzymes are unable to utilize mediators. Cass et al. investigated 11 different enzymes and suggested that Pyruvate oxidase, Sarcosine oxidase, Oxalate oxidase, Choline oxidase, lipoamide dehydrogenase, alcohol dehydrogenase work at least 10 times less efficiently compared to glucose oxidase using a ferrocinium ion as an electron acceptor. The study notes “glucose oxidase used a variety of electron acceptors,” whereas “some (oxidases) were reported to use oxygen specifically.” (Cass et al., J. Eletroanal. Chem. 190, 1985, 117-127) making them less useful for mediated electron acceptors. Ramanavicius et al. (Anal. Bioanal. Chem., 387, 1899, 2007) reported that by replacing oxygen with ferrocyanide ions, a creatine sensor was capable of design that exhibited better sensitivity, accuracy and less interference. However, the sensor is sensitive to oxygen concentration in the sample. All the measurements have to be operated in oxygen-free conditions (<5 μM of O2) using native sarcosine oxidase enzyme.
Accordingly, previous research suffered from mediator selection and the oxygen concentration in the sample using native enzymes for enzyme electrode design. The need therefore exists for biosensor designs that provide a reduced oxygen interference and sensitivity for oxidase based sensors.